The isolation of gene's whose expression differs between two cell or tissue types, or between cells or tissues exposed to chemical compounds or pathogens, is critical to understanding the mechanisms which underlie various physiological disorders. Changes in gene expression play a central role in many critical, therapeutically relevant processes including embryogenesis, aging, tissue repair and neoplastic transformation.
Several methods have been utilized for the detection and isolation of genes which are activated or repressed in response to developmental, physiological or pharmacological events. One of these methods, subtractive hybridization, is a particularly useful method for selectively cloning sequences present in one DNA population but absent in another. This selective cloning is accomplished by generating single stranded cDNA libraries from both control tissue (driver cDNA) and tissue during or after a specific change or response being studied (tester cDNA). The two cDNA libraries are denatured and hybridized to each other, resulting in duplex formation between the driver and tester cDNA strands if a particular sequence is common to both cDNA populations. Since the common sequences are removed, the remaining non-hybridized single-stranded DNA is enriched in sequences present in the experimental cell or tissue which is related to the particular change or event being studied (Davis et al., (1987) Cell, 51:987-1000).
Subtractive hybridization has led to the discovery of many important genes including the myogenesis differentiation marker MyoD1, the T-cell receptor and genes activated at the gastrulation stage of Xenopus laevis (Davis et al., 1987; Hedrick et al., (1984), Nature, 308:149-153; Sargent et al., (1983) Science, 222:135-139).
The power of the subtractive library method has been significantly enhanced by the polymerase chain reaction (PCR), which allows performance of multiple cycles of hybridization using small amounts of starting material (Wieland et al. (1990) Proc. Natl. Acad. Sci. USA, 87:2720-2724; Wang et al. (1991) Proc. Natl. Acad. Sci. USA, 88:11505-11509; Cecchini et al., (1993) Nucleic Acids Res., 21:5742-5747) .
PCR-driven subtraction hybridization using biotinylated control DNA has also been performed to identify differentially expressed genes (Lebeau et al., (1991) Nucleic Acids Res., 19:4778; Duguid et al., (1990) Nucleic Acids Res., 18:2789-2792). Duguid et al. ligated a duplex oligonucleotide referred to as an oligovector (also called a linker primer) to double stranded cDNA isolated from either control or scrapie-infected hamster brain, then digested the ligated DNA with a restriction enzyme to cleave the oligovector and reduce the amplification potential of the control DNA. The sequences were amplified by PCR and subtraction hybridization was performed to enrich for sequences present in infected brain, but absent in uninfected brain. DNA isolated from normal brain was biotinylated, mixed with DNA from infected brain, denatured and hybridized to normal DNA, and biotinylated complexes were removed. The subtracted DNA was then subjected to further subtraction/amplification cycles.
There are a number of problems associated with the existing PCR-directed subtraction hybridization methods. First, preventing amplification of the control cDNA by restriction enzyme digestion of its linker primer is often inefficient and unreliable (Kaufman et al., (1990) Biotechniques, 9:304-306). Second, since the biotinylation reaction does not proceed to completion, the subtracted cDNA is often contaminated with control cDNA which is present in the initial hybridization mixture in large excess. Moreover, the method for separating biotinylated and unbiotinylated molecules is tedious and large amounts of cDNA and expensive reagents are required. In addition, the contamination by control cDNA may necessitate an additional screening step prior to the final selection of differentially expressed genes.
There is a need for a rapid, low cost, simple, reliable, reproducible, PCR-directed subtraction hybridization method for identification of clinically and therapeutically relevant differentially expressed genes which will overcome the inherent problems associated with the prior art methods. The present invention provides such a method.